EP3588073A1 - Procédé électrochimique enzymatique pour la quantification d'analytes dans des échantillons de fluides biologiques - Google Patents

Procédé électrochimique enzymatique pour la quantification d'analytes dans des échantillons de fluides biologiques Download PDF

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Publication number
EP3588073A1
EP3588073A1 EP18382459.8A EP18382459A EP3588073A1 EP 3588073 A1 EP3588073 A1 EP 3588073A1 EP 18382459 A EP18382459 A EP 18382459A EP 3588073 A1 EP3588073 A1 EP 3588073A1
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EP
European Patent Office
Prior art keywords
dehydrogenase
enzyme
strip
electrode
biosensor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP18382459.8A
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German (de)
English (en)
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EP3588073B1 (fr
EP3588073C0 (fr
Inventor
Laura GONZÁLEZ-MACIA
Maitane URIEN BERRIO
Roberto González Rioja
Arrate JAUREGUIBEITIA CAYROLS
Israel SÁNCHEZ MORENO
Carmen HERMIDA DÍAZ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biolan Health SL
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Osasen Sensores SL
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Publication date
Priority to ES18382459T priority Critical patent/ES2980854T3/es
Application filed by Osasen Sensores SL filed Critical Osasen Sensores SL
Priority to EP18382459.8A priority patent/EP3588073B1/fr
Priority to PCT/EP2019/065994 priority patent/WO2019243314A1/fr
Priority to BR112020026107-6A priority patent/BR112020026107A2/pt
Priority to MX2020014093A priority patent/MX2020014093A/es
Publication of EP3588073A1 publication Critical patent/EP3588073A1/fr
Priority to US17/126,971 priority patent/US11898191B2/en
Priority to CONC2020/0016051A priority patent/CO2020016051A2/es
Application granted granted Critical
Publication of EP3588073B1 publication Critical patent/EP3588073B1/fr
Publication of EP3588073C0 publication Critical patent/EP3588073C0/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3274—Corrective measures, e.g. error detection, compensation for temperature or hematocrit, calibration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)

Definitions

  • the present invention refers to an enzymatic electrochemical method to determine and quantify analytes in physiological samples, which is based on the differential measurement of current intensities provided by two biosensor strips containing enzymes and mediators involved in a redox reaction of the analyte to be determined. Therefore, the invention could come under the field of analytical methods.
  • Analyte detection and quantification in physiological samples to directly or indirectly determine a pathology is highly important for today's society, and a great variety of methods and devices therefore exists to detect analytes of interest such as glucose, cholesterol, uric acid, etc.
  • a standard strategy for these detections is to apply electrochemical reactions, particularly reactions where the analyte is an enzyme substrate and where this interaction triggers a flow of electrons that can be directly related to the concentration of the analyte of interest.
  • Documents such as US2016/0177365 , US2010/0270175 or EP1770396 describe methods and sensors based on these processes, although these methods are applied to specific analytes in a limited detection range.
  • the present invention refers to an enzymatic electrochemical method to determine and quantify analytes in physiological samples which is based on the differential measurement of current intensities measured at ambient temperature between a biosensor strip A (usually referred to as blank ) modified with an enzyme and a mediator, and a biosensor strip B (referred to as complete ) which contains a cofactor in addition to the enzyme and the mediator. and its conversion to analyte concentrations performed using the calibration parameters of the corresponding biosensor strip batch.
  • the measurement method is based on the amperometric electrochemical technique, where a constant potential is applied during a specific time and the current produced by the electrochemical reaction in the cell at that time is recorded.
  • the conversion from current to analyte concentration occurs by means of a previously established calibration curve, obtained with the measurement device and biosensor strips from the same batch.
  • the sample is analysed in parallel using two different biosensor strips (blank and complete). The results obtained with both strips are subtracted, thus eliminating any interference effect and isolating the signal corresponding only to the analyte determination.
  • Each biosensor strip batch is previously calibrated using the corresponding "synthetic biological fluid" sample doped with known concentrations of the analyte to be determined in the range of interest, which are electrochemically measured by amperometry. Calibration codes are thus generated and subsequently used to calculate the analyte concentrations in real samples.
  • the "synthetic biological fluid” is a solution that contains the main interferences of the biological sample and whose electrochemical behaviour is close to the real matrix. Its use together with the dual biosensor strip system enables the determination of low concentrations of analyte in real biological fluids without the need of sample pre-treatment.
  • a biosensor strip consists of at least three screen-printed electrodes: a reference electrode, which may be Ag/AgCI, Ag, Pt, Au; an auxiliary electrode (or counter electrode), which may be graphite, carbon, Pt, Au; and a working electrode, which may be carbon, graphite, graphene, Au.
  • the screen-printed electrodes are deposited on a substrate, preferably PET, ceramic or paper.
  • a layer of isolating ink is screen-printed on top of the electrodes, protecting them from direct contact with the solution and defining the working area.
  • the enzyme, the mediator, the cofactor and other components of the modification, if applicable, are deposited on the working electrode within each test strip.
  • the applied potential in this invention ranges between -0.2 V and +0.2 V vs. Ag/AgCI.
  • the current obtained for each analyte concentration vs. the known concentration of that analyte provides the calibration curve for all the biosensor strips produced in the same batch.
  • the present invention relates to an enzymatic electrochemical method to quantify analytes in isolated biological fluid samples that comprise the following stages:
  • the enzyme present on the working electrode of the test strips is an oxidoreductase.
  • the enzyme is a dehydrogenase, which optionally may be NAD+ or NADP+ dependent.
  • the dehydrogenase enzyme is selected from among lactate dehydrogenase, glucose dehydrogenase, methanol dehydrogenase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, glycerol-3-phosphate dehydrogenase, glycerol dehydrogenase, alcohol dehydrogenase, D-xylose-1-dehydrogenase.
  • the mediator present on the working electrode of the biosensor strips is selected from among tetramethylbenzidine (TMB), potassium ferricyanide, toluidine blue, hydroquinone, tetrathiafulvalene (TTF), ferrocene.
  • TMB tetramethylbenzidine
  • TTF tetrathiafulvalene
  • the cofactor present on the working electrode of the biosensor strip B is selected from among NAD+, NADP+, FAD, FMN.
  • the cofactor does not form part of the structure of the enzyme.
  • the applied potential in stage (b) is between -0.2 V y +0.2V vs. Ag/AgCI. In a more preferred embodiment, the applied potential in stage (b) is between -0.05 V and +0.15 V vs. Ag/AgCI.
  • the time during which the potential is applied in stage (b) is between 20 and 120 s. In a more preferred embodiment, the time during which the potential is applied is stage (b) is between 30 and 90 s.
  • the fluid sample is an isolated biological sample that is selected from among urine, blood, serum, plasma, pleural fluid or saliva.
  • the enzyme present on the electrodes is preferably an oxidoreductase, more preferably a dehydrogenase, and even more preferably a dehydrogenase which preferably may be dependent on cofactors such as NAD+ or NADP+.
  • the dehydrogenase enzyme is selected from among lactate dehydrogenase, glucose dehydrogenase, methanol dehydrogenase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, glycerol-3-phosphate dehydrogenase, glycerol dehydrogenase, alcohol dehydrogenase, D-xylose-1-dehydrogenase.
  • a “mediator” is a compound with redox activity that facilitates the electron transfer between the cofactor and the surface of the working electrodes of each test strip, lowering the applied potential (in absolute value) that can be used to determine the analyte.
  • the mediator present in the electrodes is selected from among tetramethylbenzidine (TMB), potassium ferricyanide, toluidine blue, hydroquinone, tetrathiafulvalene (TTF), ferrocene.
  • the cofactor is selected from among FAD, FMN, NAD+, NADP + .
  • the cofactor is external and it does not form part of the structure of the enzyme.
  • the method of the invention is applicable to any biological fluid sample, although preferably, the isolated biological sample is selected from among urine, blood, serum, plasma, pleural fluid or saliva, and more preferably, urine.
  • the described calibration stage is conducted using analyte solutions in samples of synthetic biological fluids.
  • an analyte standard prepared in synthetic biological fluid sample is used and it is progressively diluted in the same synthetic sample to obtain the samples for the calibration.
  • these concentrations vary depending on the analyte and each measurement is conducted at least in triplicate.
  • a "synthetic biological fluid sample” is understood as an artificial matrix that presents similar characteristics to the natural biological sample (urine, blood, plasma, etc.) and which is obtained by adding the components that it typically comprises of, such as salts, proteins, sugars, etc.
  • the invention will be next illustrated by some assays performed by the inventors, showing the effectiveness of the method of the invention.
  • Example 1 D-xylose determination in different media
  • biosensor strips contain D-xylose dehydrogenase as a biological recognition element, NAD+ as an enzyme co-factor and toluidine blue (TBO) as a mediator.
  • the buffer is 200 mM phosphate buffer pH 8 and the synthetic urine contains 150 mM KCI and 10 g/L urea.
  • Figure 1 shows the electrical current in nanoamperes obtained in buffer, synthetic urine and human urine samples with varying concentrations of D-xylose, when measured at 0 V.
  • the D-xylose biosensor strip of the present invention has a linear response respect to D-xylose concentration in the range from about 0,75 mg/dL to about 15 mg/dL, in various media.
  • the calibration curve in buffer was quite different from the calibration curve in human urine samples.
  • the calibration curve in synthetic urine is quite similar to the calibration curve in human urine samples, and it can be used for the highly accurate determination of D-xylose in human urine (see Figure 1 ).
  • Example 2 Accuracy of the method of the invention for the determination of D-xylose concentration in human urine samples
  • the method consists of two biosensor strips: the blank strip, containing TBO and D-xylose dehydrogenase (XDH) and the complete strip, containing TBO, D-XDH and NAD+.
  • Table 1 shows the electrical currents in nanoamperes of several human urine samples containing different D-xylose concentrations and the % accuracy respect to the real D-xylose concentration.
  • Urine sample 1 Urine sample 2
  • Urine sample 3 [D-xylose]/ mg*dL-1 i / nA %recovery i / nA %recovery i / nA %recovery 0.75 134.8 112.9 119.7 94.3 121.1 96.1 1.25 181.7 102.3 175.3 97.6 178.9 100.2 2.0 275.8 107.3 256.0 98.2 260.0 100.0 3.75 449.1 99.8 431.5 95.5 415.9 91.7 15 1462.0 87.2 1572.3 94.0 1403.7 83.6
  • D-xylose determination in human urine samples was performed using a mono-strip biosensor system and the bi-strip biosensor system of the invention.
  • FIG. 3 shows the recovery percentage of D-xylose expressed as the ratio of concentration levels obtained using the biosensor strips of the invention respect to real concentration levels, by applying only one complete strip or the bi-strip biosensor system above proposed in the invention. As it can be observed, this system improved remarkably the accuracy of the sensor system, especially at low analyte concentrations.

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  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
EP18382459.8A 2018-06-21 2018-06-21 Procédé électrochimique enzymatique pour la quantification d'analytes dans des échantillons de fluides biologiques Active EP3588073B1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
EP18382459.8A EP3588073B1 (fr) 2018-06-21 2018-06-21 Procédé électrochimique enzymatique pour la quantification d'analytes dans des échantillons de fluides biologiques
ES18382459T ES2980854T3 (es) 2018-06-21 2018-06-21 Método electroquímico enzimático para la cuantificación de analitos en muestras de fluidos biológicos
BR112020026107-6A BR112020026107A2 (pt) 2018-06-21 2019-06-18 Método eletroquímico enzimático para quantificação de analitos em amostras isoladas de fluido biológico
MX2020014093A MX2020014093A (es) 2018-06-21 2019-06-18 Metodo electroquimico enzimatico para la cuantificacion de analitos en muestras de fluidos biologicos.
PCT/EP2019/065994 WO2019243314A1 (fr) 2018-06-21 2019-06-18 Procédé électrochimique enzymatique pour la quantification d'analytes dans des échantillons de fluides biologiques
US17/126,971 US11898191B2 (en) 2018-06-21 2020-12-18 Enzymatic electrochemical method for the quantification of analytes in biological fluid samples
CONC2020/0016051A CO2020016051A2 (es) 2018-06-21 2020-12-21 Método electroquímico enzimático para la cuantificación de analitos en muestras de fluidos biológicos

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EP18382459.8A EP3588073B1 (fr) 2018-06-21 2018-06-21 Procédé électrochimique enzymatique pour la quantification d'analytes dans des échantillons de fluides biologiques

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EP3588073A1 true EP3588073A1 (fr) 2020-01-01
EP3588073B1 EP3588073B1 (fr) 2024-05-22
EP3588073C0 EP3588073C0 (fr) 2024-05-22

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US (1) US11898191B2 (fr)
EP (1) EP3588073B1 (fr)
BR (1) BR112020026107A2 (fr)
CO (1) CO2020016051A2 (fr)
ES (1) ES2980854T3 (fr)
MX (1) MX2020014093A (fr)
WO (1) WO2019243314A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2972404B1 (fr) 2013-03-15 2021-11-24 Abbott Laboratories Analyseurs diagnostiques automatiques comprenant des systèmes de rail accessibles par l'arrière et procédés associés

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6736957B1 (en) * 1997-10-16 2004-05-18 Abbott Laboratories Biosensor electrode mediators for regeneration of cofactors and process for using
EP1770396A2 (fr) 2005-09-30 2007-04-04 Lifescan, Inc. Procédé et appareil d'analyse électrochimique rapide
US20090018424A1 (en) * 2006-10-04 2009-01-15 Dexcom, Inc. Analyte sensor
US20100270175A1 (en) 2009-04-22 2010-10-28 Nova Biomedical Corporation Electrochemical biosensors based on NAD(P)-dependent dehydrogenase enzymes
US20160177365A1 (en) 2013-08-07 2016-06-23 Arkray, Inc. Substance Measurement Method and Measurement Device Employing Electrochemical Biosensor

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6736957B1 (en) * 1997-10-16 2004-05-18 Abbott Laboratories Biosensor electrode mediators for regeneration of cofactors and process for using
EP1770396A2 (fr) 2005-09-30 2007-04-04 Lifescan, Inc. Procédé et appareil d'analyse électrochimique rapide
US20090018424A1 (en) * 2006-10-04 2009-01-15 Dexcom, Inc. Analyte sensor
US20100270175A1 (en) 2009-04-22 2010-10-28 Nova Biomedical Corporation Electrochemical biosensors based on NAD(P)-dependent dehydrogenase enzymes
US20160177365A1 (en) 2013-08-07 2016-06-23 Arkray, Inc. Substance Measurement Method and Measurement Device Employing Electrochemical Biosensor

Also Published As

Publication number Publication date
EP3588073B1 (fr) 2024-05-22
BR112020026107A2 (pt) 2021-03-16
MX2020014093A (es) 2022-02-10
ES2980854T3 (es) 2024-10-03
US20210102232A1 (en) 2021-04-08
US11898191B2 (en) 2024-02-13
WO2019243314A1 (fr) 2019-12-26
CO2020016051A2 (es) 2021-02-08
EP3588073C0 (fr) 2024-05-22

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